IE57797B1 - Externalization of products of bacteria - Google Patents

Abstract

A bacterial product is made by transforming a temperature sensitive lysogen with a DNA molecule which codes, directly or indirectly, for the product, culturing the transformant under permissive conditions and externalizing the product by raising the temperature to induce phage encoded functions.

Description

COMPLETE SPECIFICATION EXTERNALIZATION OF PRODUCTS OF BACTERIA.

APPLJCAT now SPECIFICATION F5L5D Λ SMITHKLINE BECKMAN CORPORATION, a corporation organized under the laws of the Commonwealth of Pennsylvania, one of the United States of America, of One Franklin Plaza, Philadelphia, Pennsylvania 19103, United States of America. - 1 This invention, relates to genetic engineering and, in particular, to externalisetion of products produced by genetically engineered microorganisms.

A problem with using S, coli and other prokaryotic microorganisms as hosts for expression of desired proteins has often been externalising the proteins from the host cells for purification. Attempts to overcome this problem include physical disruption of cells such as by homogenisation or sonication, chemical disruption of cells such as by treatment with detergent or lysozyme, and fusing a DMA sequence which codes for an excretion signal peptide to a structural gene coding for the desired product. For example, Weissman et al„, European Patent Application 61,250, disclose treatment of host cells with a lysing or permeabilising agent; Silhavy et al.? u.S, Patent 4,336,336, disclose a method for fusing a gene for a cytoplasmic protein to a gene for a non-cytoplasmic protein, so that a resulting hybrid - 2 protein is transported to, near or beyond the host cell surface; Gilbert et al., U.S. Patent 4,338,397, disclose a method for producing mature secreted proteins comprising inserting a structural gene for a preprotein into an expression vector.

E. coli can be infected by an obligatory parasite, the lambda phage, which is a double-stranded DNA virus. Lambda genetics, like E. coli genetics, is well-studied. See, for example, ’The Bacteriophage Lambda,™ edit, by A.D. Hershey, Cold Spring Harbor Laboratory, New York, 1971.

Lambda, a temperate phage, multiplies in E. coli in either of two phases. In one, the lytic phase, the phage DNA replicates autonomously and directs formation of capsid proteins, packaging and host cell lysis.

Expression of lambda DNA during the lytic phase is highly efficient. Transcription occurs on both DNA strands, on one in the rightward direction and on the other in the leftward direction. Induction can result in release of about one hundred phage particles within 50 minutes at 37°C. See, Hershey, above.

In the other phase, the lysogenic phase, lambda DMA is integrated into the host cell genome and is replicated, passively, along with the host chromosomal DNA by the host replication enzymes. A phage in the lysogenic phase is known as a prophage; the host is known as a lysogen and is said to be immune.

Immunity can be lost by occurrence of various events which induce the lytic phase. The products of the lambda int and xis genes catalyse excision of the lambda genome from the E. coli genome to form a covalently closed - 3 circle capable of autonomous replication. The synthesis of these genes r and either directly or indirectly,, all other lambda genes is repressed by the product of the lambda cl gene. In response to certain chemicals or DNA-damaging agents, the bacteria directs synthesis of the product of the bacterial recA gene. The recA gene product proteolyticallv cleaves the cl repressor protein, permitting expression of the lytic phase genes.

Propagation of the phage then requires interplay of several lambda regulatory elements which ultimately initiate autonomous replication of the lambda DNA. The products of the lambda P and 0 genes are required for DNA replication. Subsequent to DNA replication the phage must direct synthesis of viral structural proteins, that is, head and tail proteins, and their assembly into intact empty virions. Interaction of at least 18 genes is required to accomplish this. Finally, the DNA is packaged into the empty virions to produce infectious intact virions, and the cell is ruptured by endolysin, coded for by the lambda S and R genes which are activated by the product of the Q gene, thereby releasing the phages. The Q gene is activated by the N function. The N gene is repressed by the cl function.

The 18 genes required for capsid assembly lie between about map positions 3 and 36 on the rightward transcription strand, map positions being representative of percentages of total lambda DNA. The first genes, from left to right, are A, W and 3; the last is J. In normal lysogens, to the right of the J gene are eight bacterial genes. Five of these, bio A, 8, C, D and F, are involved in biosynthesis of biotin. A sixth, uvr8, confers resistance to ultraviolet radiation. The final two, chiA and E, confer sensitivity to chlorates. See, Guest, Mol. Gen. Genet. 105: 285-289 (1969) and Stevens et al, in aThe - 4 Bacteriophage Lambda, eel. by Hershey, et al, cited above, at pp. 515-53 4.

Another lambda gene which functions in natural host cell lysis is the kil gene. The function of the kil gene is not fully understood. Cells which express the kil gene have a decreased rate of cell growth following induction. Loss of the kil function permits cells to grow at normal rates, that is, log phase growth, after induction, until lysis occurs. Like the S and R genes, the kil gene is regulated by the cl repressor,, indirectly, through the N gene. See, Greer, Virology 66:589-604 (1975).

Temperature sensitive lysogens have been well-studied. They are described, for example, by Campbell, Virology 14: 22-32 (1961). The cl857 gene is a temperature sensitive cl mutant. It is functional at or below 38°C. See, Sussman et al., C.R.H- Acad. Sci.

Paris 254:1517-1519 (1962). Similar phage systems are known to occur in other genera. For example, Lomovskaya et al., J. Virol. 9.-258-262 (1972), report temperature sensitive mutants of a temperate phage which infects Streptomyces; Flock, Mol. Gen. Genet. 155: 241-247 (1977) , reports temperature sensitive mutants of the temperate phage- phi-105, which infects Bacillus; Botstein et al., Nature 251: 584-588 (1974) report temperature sensitive mutants of the temperate phage, P22, which infects Salmonella. Jostrom et al., J. Bacterio!. 119:19-32 (1974), and Thompson, J. Bacteriol. 129:778-788 (1977), report temperature sensitive mutants of the temperate phage, phi-11, which infects Staphylococcus; Miller et al, Virol. 59:566-569 (1974) report temperate phages of Pseudomonas. - 5 The lambda endolysin has been found to lyse Salmonella strains which are able to absorb the phage as reported by Botstein et al. t Ann,,, Rev. Genetics 16:51-83 (1982) .

Perricaudet et al.? FEES Lett. 56:7-11 (1975), describe deletion of lambda genes between map positions 58 and 71 ( Δ58-71) which segment includes the lambda int? xis, red? gam? dll and kil genes.

Hershberger et al.? European Patent Application 2?084?584? disclose use of a lysogen as a host cell to stabilize and select for the presence of a plasmid. The authors disclose? for example? transforming a lysogen having a defective cl gene with a plasmid carrying a functional cl gene. In one disclosed embodiment, the functional cl gene is the cl857 gene.

It is known that transposable elements? that is, genes which can recombine independently of host chromosomal recombination mechanisms? can be inserted into host cells as markers. Ross et al.. Cell 16:721-731 (1979), report physical structures of deletions and inversions promoted by the transposable tetracycline-resistance element? tnlO. Davis et al.? Bacterial Genetics? Cold Spring Harbor Laboratory? New York (1980)? describe uses of transposable elements.

Ruvkun et al.? Nature 289:85-88 (1981)? report integration of the transposable kanamycin resistance and neomycin resistance element, tn5, into Rhizobium meliloti chromosomal DNA by conjugation of a plasmid carrying tn5 followed by homologous recombination. Integration of a heterologous gene by recombination resulting from presence of homologous flanking sequences is also disclosed in - 6 European Patent Application No» 74s808.

The invention is a method of producing a product in bacteria utilising endolysin-encoding genes from temperate phages» The method comprises transforming a temperature sensitive bacterial strain, which carries a temperature sensitive phage repressor gene and functional phage lysozyme-encoding genes such that the lysozyme-encoding genes are repressed under permissive conditions and expressed under restrictive conditions, with a DNA molecule(s) which expresses, directly or indirectly, the product; culturing the transformed strain under permissive conditions such that the product is made? raising the temperature to produce restrictive conditions; and, optionally, recovering the product from the culture medium or a concentrate thereof.

Another aspect of the invention is a method of producing a product in a bacteria which comprises transforming a bacteria which produces the product with a phage ONA sequence which carries a temperature sensitive phage repressor gene and phage lysozyme-encoding genes such that the lysozyme-encoding genes are repressed under permissive conditions and expressed under restrictive conditions, to make the bacteria lytic; culturing the transformed bacteria under permissive conditions such that the product is made; changing the temperature to provide restrictive conditions; and, optionally, recovering the product from the culture medium or a concentrate thereof.

Another aspect of the invention is a ONA fragment comprising a defective phage sequence having a temperature sensitive repressor gene and functional lysozyme-encoding genes such that the lysozyme-encoding genes are repressed under - 7 permissive conditions and expressed under restrictive conditions, a selectable marker and, preferably, flanking DNA sequences which are homologous to a contiguous sequence in the chromosome of a host cell.

Other aspects of the invention are a method of making a lytic bacteria which comprises transforming a bacteria with the DNA fragment of the invention, and bacteria comprising said DNA fragment.

' Yet another aspect of the invention is a method of administering a product to a mammal comprising administering an amount of a temperature sensitive bacteria containing an effective dose of the product to the mammal, whereby the bacteria lyse within the mammal and release the product.

A temperature sensitive bacteria is one which carries a prophage DNA sequence including a temperature sensitive repressor gene such that when cultured at one temperature range (permissive conditions) the repressor is functional but when cultured at another temperature range (restrictive conditions) the repressor is not functional; the repressor is not expressed or is not stable. Under restrictive conditions, the phage genes, including phage lysozyme-encoding genes, are expressed leading to cell lysis. Such temperature sensitive bacteria are lytic bacteria. Any lytic bacteria, as herein defined, can be used in the method of the invention.

Temperature sensitive temperate phage repressor genes are available or can be made by mutating such genes by procedures known to the art. By way of example, - 8 Campbell, virology 14:22-32 (1961), describes a procedure for isolating temperature sensitive phage mutants. Generally, the procedure comprises mutagenizing phage-infected bacteria, such as by ultra-violet irradiation, and then incubating survivors at high temperature to cause induction of any temperature sensitive repressor mutants. The lysate is then used to infect sensitive bacteria. The new lysogens are subjected to heat induction and phage produced following the heat induction are used to prepare lysogens from sensitive bacteria. This cycling (Ivsogen preparation, induction, re-preparation) leads to identification, of phage repressor mutants. Typically, 3 to 4 such cycles are sufficient to yield such mutants.

The description which follows relates, in large part, to lytic E. coli and, especially, to cl857 E. coli lysogens. Nevertheless, from said description persons of ordinary skill in the art will be enabled to practice the invention as it relates to other lytic E. coli as well as to other lytic bacteria, using repressor and endolysin-encoding genes from lambda or from other temperate phages, such as the temperate phages noted above. £1857 lysogens, which are known and commonly available, produce cl repressor which is active at or below 38°C but inactive above 38°C. These are preferred over other temperature sensitive E. coli lysogens because in addition to a mutation rendering the repressor inactive above 38°C, the cl857 gene contains a second mutation which causes the cl repressor protein to be insensitive to proteolytic cleavage by the product of the lambda recA gene. Thus, when cultured under permissive conditions, the cl repressor protein is stable and, therefore, effective in maintaining immunity. - 9 _ E. coli strain UC5822 is a lysogen which has the cI857 mutation. It also has a point mutation in the int gene (int 6 am, an amber mutation) and in the j? gene (P3 am, an amber mutation). (Amber mutations signal' termination of translation). UC5822 is generally preferred over, for example, MM294(cI857) because UC5822 is defective, that is, it does not generally produce infectious phage particles. Defective lysogens are preferred, especially when used to administer a polypeptide to a mammal. UC5822, however, produces lower levels of the lysozyme, presumably because it has a lower copy number of the S and R genes, namely, one, than does MM294(cl 857), namely, fifty to one hundred, after induction. Nevertheless, UC5822 lyses readily following induction.

In one aspect of the method of the invention, temperature sensitive bacteria are transformed with a DNA molecule (s) which codes, directly or indirectly, for a desired product. The transformation may be carried out by any technique which allows the DNA molecule to enter the host cell and to express the product. Techniques include, for example, transformation, transduction, conjugation and cell fusion. Many suitable expression vectors are well known and publicly available as are techniques for cloning genes for products and transforming cells with such molecules. Generally, the product will be a non-excreted, heterologous gene product, that is, one which is not naturally produced by the host and which is not externalized. Products which are expressed directly include polypeptides; products which are expressed indirectly include polypeptides, glycoproteins, antibiotics and other molecules such as, for example, metal ions which can be sequestered within a metallothionein-producing bacteria. - 10 Transformed host cl 857 lysogens can he grown up indefinitely under permissive conditions (-38°C, usually 32 to 36°C) which are optimal for expression of the desired product. When sufficient growth has been achieved, that is, usually, when mid-log phase growth (A-cn ~ θ·5) has been achieved, synthesis of lambda endolysin is induced by culturing the lysogen under restrictive conditions., This can be accomplished by raising the temperature of the culture medium, or of a cell concentrate thereof, to, in the case of cl 857, greater than 38°C, preferably 42 to 44°C, for about 90 to 120 minutes. Alternatively, the temperature of the culture medium, or of a concentrate thereof, is raised to greater than 38°C, preferably 42 to 44°C, for a shorter time, that is, a time sufficient to induce the phage DNA, preferably at least about five minutes, following which the temperature is lowered to 0 to 38°C, preferably 2 to 35°C.

Maintaining restrictive conditions for 90 to 120 minutes is preferred because lysis is more efficient and rapid. However, the latter procedure is preferred in certain applications, for example, when a desired protein is heat labile or when the cost of maintaining the restrictive conditions is prohibitive.

If the host £1857 lysogen has a functional lambda cro gene, the cells will continue to synthesise the lysozyme at any temperature at which the cells function, until lysis occurs. Although lambda endolysin is active as low as O°C, the time needed for lysis is longer at low temperature due to a decrease in rates of protein synthesis and catalytic activity generally.

Just prior to or following induction, the cells are preferably concentrated, such as by filtration, centrifugation or other means, and incubated in this concentrated form until lysis. Such procedure facilitates collection and purification of the desired product. Following induction, the bacterial cell wall is substantially degraded. The cells, in the form of protoplasts, will continue to synthesize the desired product which is largely released into the medium through the cell membranes» Complete release into the medium is effected by lysis. Lysis is observable as a clarification of the culture medium or concentrate and/or an increase in the viscosity of the culture medium or concentrate. Lysis can be enhanced such as by mechanical agitation or rapidly changing the culturing conditions, for example, by rapidly changing temperature between 2 and 25°C or changing the osmotic strength of the medium or concentrate.

Preferably, after concentrating cells and decanting product-containing supernatant, induced cells are suspended in a minimal salts buffer or 0.1 M tris buffer, 50 mM NaCl and 1 raM ED TA and agitated to effect lysis.

The desired product can then be recovered from the medium or concentrate and purified, if desirable, by known techniques.

In an alternative procedure, whole cells are concentrated and administered orally to a mammal prior to induction of the lytic phase. Induction will then occur internally, resulting in release of the desired polypeptide. This method can be especially useful for administering antigens to animals in cases in which whole cells as well as the desired antigen are preferred to provoke an immunoprotective response. For example, temperature sensitive lysogens carrying genes which code for antigens such as the LT-B antigen can be fed directly - 12 to pigs and/or calves. The amount of cells administered to each animal will be that amount which contains an effective dose. The amount of protein produced by a unit amount of cells can be calculated by known techniques.

An aspect of the invention is a DNA fragment which can be used to construct a lytic bacteria for use in the method of the invention. Such DNA fragment comprises a defective phage sequence having a temperature sensitive repressor gene and functional lysozyme-encoding genes.

Such DNA fragment comprising a defective lambda sequence has a temperature sensitive cl gene and functional lambda endolysin-encoding genes (N, Q, S and R) such that the endolysin is expressed under restrictive conditions, a selectable marker, and, preferably, flanking DNA sequences which are homologous to a contiguous DNA sequence in a host cell chromosome. In one particular embodiment, the DNA fragment comprises lambda DNA which is deleted in the genes lying between map positions 58 and 71, and therefore lacks the xnt, xis and kil genes, has a temperature sensitive cl gene and has mutations in the 0 and P genes and in which the cl gene is the ££857 mutant and produces endolysin under restrictive conditions. The O and P mutations can be deletion or point mutations. Point mutations, such as the 029, P3 and P80 mutations, are preferred because they are readily available. The P3 mutation is preferred over the P80 mutation.

In another particular embodiment, the fragment comprises lambda DNA which is substantially deleted in the genes lying between map positions 3 and 71. Such fragment lacks substantially all genes essential for lambda capsid assembly as well as the int, xis and kil genes.

A host cell, Ξ. coli or other bacteria, which produces a desired product? or which is previously or subsequently made to produce the desired product? such as by genetic engineering techniques, can be transformed with a phage DNA sequence which carries temperature sensitive phage repressor gene and phage lysozyme-encoding genes by known techniques. These include infecting the bacteria with a temperate phage having such temperature sensitive repressor gene? preferably a defective phage. These also include transforming the bacteria with the DNA fragment of the invention by known techniques, for example, transformation, transduction, conjugation and fusion. Transformation generally involves incorporating the fragment into a vector, such as a phage or a plasmid. For example, the fragment can be cloned into a plasmid, such as pBR322 or others, and grown up in vivo in an appropriate a host which is lacking a contiguous sequence homologous to sequences flanking the fragment or which is defective for recombination events (rec )» The plasmid can be recovered and used to transform an appropriate host for production of a desired product. Following transformation of such host, the fragment which has flanking DNA sequences homologous to a contiguous DNA sequence in the host chromosome will integrate by spontaneously recombining at the site of the homologous contiguous sequence. Alternatively, an appropriate host for production of a desired product can be transformed with the isolated DNA fragment in linear or circular form.

The DNA fragment carries a selection marker to facilitate selection of transformants. Selectable markers are typically genes which code for assayable enzymes, which restore prototrophv to an auxotrophic host or which confer resistance to lethal or inhibitory compounds, usually antibiotics. Preferably, the selection marker is a gene which confers antibiotic resistance as these do not requite use of an auxotrophic host which may not be available or which can spontaneously revert to prototrophy- Tetracycline resistance is preferred because tetracycline is inexpensive and because resistance to tetracycline is not normally spontaneously acquired.

Presence of the marker in transformants indicates that the host comprises the DNA fragment. If the fragment integrates, the whole fragment will integrate because homology between the DNA fragment and the host cell DMA exists only in regions flanking the lambda DNA and the marker.

Absent a marker in the DNA fragment, selection of host cells carrying the lambda DNA following transduction or other transforming procedure would require superinfecting putative transducfcants with a defective phage (non-integrating) and selecting for immune bacterial survivors.

E. coli strains made lytic by integration of a DNA fragment of the invention include, for example, MG strains. These strains are lysogens in which the lambda DNA is deleted in genes lying between map positions 58 and 71, and therefore lack the int, xis, red, gam, kil and dll genes, has the cI857 mutation and has mutations in the 0 and P genes and has functional N, Q, S and R genes such that endolysin is expressed under restrictive conditions. In one embodiment, strain MG1 [C600 (λ Δ 58-71, cl 857, P3, 029), SuII*, galK, lac2, thi, gal;:tnlO tet ], the point (amber) mutation, is read through and the O and P genes are expressed because of the production by the host cell DNA of an amber suppressor, that is, a. translational suppressor of the UAG translation termination codon. - 15 A more preferred host cell for use in the method of the invention is one which is phenotypically O and p’. One embodiment? strain MG3 [M99 (λ Δ 58-71, c!857, P3, 029) galK, lac2, fchi, gal;: fcnlO tetR] , carries the same lambda DMA fragment as strain MG1. However, it is — — R phenotypically 0 and P as well as tet , Δ k il, Δ int and Δ xis.

The most preferred lytic E. coli are MG4 strains. These are strains which are deleted in substantially all of the lambda structural protein and assembly genes and the normal right prophage-bacterial R junction, that is, the right attachment site (att ). In particular, they are lysogens which are deleted in substantially all lambda genes lying between map positions and 71, have point mutations in the O and P genes, have a temperature sensitive cl gene and have functional N, Q, S and R genes such that endolysin is expressed under restrictive conditions, and have a selectable marker, namely, the tnlO tetracycline resistance transposable element. Such defective lysogens have 4 independent blocks to viral propagation; (i) loss of 0 and P R replication functions, (ii) loss of att which renders the prophage incapable of being complemented by int and xis genes from a super infecting phage, (iii) inability to encode lambda structural genes and, (iv) the size of the lambda DMA is far below the minimum size required for packaging. These can be initially prepared by chlorate-stressing cl 857, Ο , P lysogenic strains, such as MG strains, to produce chlorate resistant mutants and selecting such mutants which are unable to complement propagation of a super infecting heteroimmune or virulent lambda or lambdoid phage deficient in A and B functions.

A DMA fragment comprising the marker, the lambda DMA and flanking sequences from the E. coli chromosome can be - 16 isolated from MG4 strains such as by treatment with restriction endonucleases or Pl transduction.

HG4 can be derived from MG3 by deleting all or most of the bacteriophage genes which encode the viral structural components. In order to verify the loss of these genes it is sufficient to demonstrate that the viral genome in MG4 is unable to complement and propagate a super infecting phage which is itself defective for these genes, λ charon 3A (λ a", B~ imrn^O) is an example of a phage which can be used for this super infection. Alternatively any phage carrying amber mutations in the A, B or other viral structural cistron can be plated on sensitive E. coli in the presence of a heteroimmune or virulent phage ( Xvir). Recombination will occur between the two phages and lead to the formation of a recombinant, for example, virA~. The frequency of recombinants will be between 1 - 50%, depending on the experimental conditions. Recombinants can be recognized by their ability to plate on suppressor containing lysogens [Y mel (λ)] and their inabililty to produce plaques on non-suppressing, λ sensitive strains, such as N99.

Plaques obtained from the above cross are plated onto petri dishes containing Y mel (λ ) or N99 and recombinants are purified. Lambda phages deficient in A, and/or 3 gene function are preferred since as a consequence of their position on the lambda genome MG4 candidates which cannot complement for these functions must lack all other lambda structural genes. The use of defective phages and hosts in this way is referred to as ’’marker rescue and is widely practiced, See, for example, 'The Bacteriophage Lambda, edit by A. D. Hershey, Cold Spring Harbor Laboratory, 1971, especially, Stevens et al., at pp. 515-533. - 17 The instant invention can be used to produce any product of bacteria. Examples are many and include, among others, insulin, rabies glycoprotein, K99 and 987? antigens, antibiotics, growth hormones, metallothioneins, alpha-1-antitrypsin, influenza antigens, lymphokines and interferon. In addition, the invention can be used in colony screening, RNA isolation and plasmid preparation, as the invention greatly simplifies and shortens the time needed for such procedures by by-passing the lysis step otherwise required.

In the following examples, which are illustrative of the invention and not limiting, all starting materials are readily available or can be readily prepared by techniques known in the art. Transductions were carried out substantially as described in ’Experiments in Molecular Genetics*, edit, by J. H. Miller, Cold Spring Harbor Laboratory, New York, (1972) pp. 201-205, which is incorporated herein by reference as though fully set forth.

Example 1 Cons truction of MGO Strain CSOO (E. coli Sull'*' K12 qalK lac_2 sull thi) was incubated in the presence of λ £1857 P3 029 (gift of W. Sysbalski, u. of Wisconsin). After overnight growth at 32°C, surviving bacteria were isolated and purified. Eighty percent of these bacteria were found to be immune to super infection, to be unable to grow at 44°C, and to produce lambda phage (following exposure to 44°C) which were indistinguishable from λ cl857 P3 029 (as judged by the ability of the phage to produce plaques on strain C600 but not on strain N99 (E. coli K12 galK XacZ suO thi) .

One of this class of survivors was purified and given the - 18 designation MGO (C500 ( λ cX857 j?3 029) ).

Example 2 Construction of MGO-&R5 Strain N5151 (EL coli K12 SA500 galK lacZ pro thr his gal8 ( λ cl857 Δ 58-71 ΔΗ1)) was incubated in the presence of PlcmlOO phage which had been grown on strain AR4 (EL coli K12 gal;;tnlO (PlcmlOO)). The cross between N5151 and PlcmlOO grown on AR4 resulted in the isolation of tetracycline resistant, UV sensitive, temperature sensitive lysogens. One of these isolates was purified and designated MGQ-AR6 (E. coli K12 ga!8 gals:tnlO λΔ 58-71 cI857 Δ Hl (bio uvrB) ) .

Example 3 Construction of M61 MGO-AR6 was made a PlcmlOO lysogen by isolating survivors of ARS which had been incubated in the presence of PlcmlOO. The PlcmlOO lysogen of MGO-ARS was designated MGQ-AR18.

Strain MQO was crossed by Pl transduction with PlcmlOO which had grown on MQO-AR18. After permitting time for phage absorption, the cells were subjected to a 9' UV fluence of 4. J/m (irradiation of 254 nm light was at a rate of 2 J/m'/s as determined by a UV dosimeter) and incubated in the presence of tetracycline. Eleven percent of tetracycline resistant colonies were resistant to UV light indicating that they did not carry the Hl deletion and thus that they possessed the lambda genes from ex through the right hand end of the phage. Specifically, this means that these clones carry the P3 and 029 mutations and intact S and R genes» One third of the UV - 19 resistant, tetracycline resistant cells were incapable of producing phage. These were therefore judged to have acquired the 58-71 deletion of lambda, and thus to have lost the int, xis and kil genes. This class was purified and designated. MG1 (C600) ( ΧΔ 58-71 cI857 P3 029) SuII+ qalK lacZ thi gal::tnlO tetR).

Example 4 Construction of MG 3 MG1 was incubated in the presence of PlcmlOO and survivors were purified. Among these survivors, a high percentage of MG1 cells which had become PlcmlOO lysogens were identified. A PlcmlOO lysogen of MG1 was purified and designated MG2.

Strain N99 was crossed by PlcmlOO transduction with PlcmlOO which had grown on MG2. Tetracycline resistant transductants were selected. All of these were found to be immune to lambda and were therefore judged to be lambda lysogens. One of these lysogens was purified and designated MG3 (N99 (ΧΔ 58-71 cl857 P3 029)).

MG3 was determined to lyse subsequent to exposure to 44°C for 90-120 minutes. No phage were found in cell cultures either prior to or after such exposure (< 1/0.1 8 ml of a culture having 3.3 x 10 cells per ml). The presence of phage was assayed on C600 cells. Control cultures of E. coli strains which harbor non-defective 5 9 lambda prophages contained between 10 and 10 phages 8 per ml of a culture having 3.3 x 10 cells per ml.

Example 5 Construction of MG3 - 20 Strain MG3 was constructed substantially as described in the above Examples except that strain N99 was lysogenized directly with λ cl857 P3 029, The resulting lysogen was crossed by Pl transduction with strain MG0~AR18 and. tetracycline resistant lysogens which lysed upon temperature induction but which did not produce phage were selected.

Example 6 Construction of UC5822 Strain UC5822 was constructed by infecting strain NS9 with λ into red3 c!857 P80 and λ hy5 climm21 Δ b2. λ hy5 climm21 Δ b2 is a hybrid between phage λ and phage 21. The purpose of λ hy5 elimm21 Δ b2 in this construction is to provide int function in tr arts which is required in order for λ into red3 cl 85 7 P80 to lysogenize this strain. The Δ b2 mutation renders the Δ hy5 climm21 Δ b2 phage incapable of directing its own integration into this strain. A survivor of this cross which displayed immunity to super infecting lambda but was sensitive to phage 21 was purified and designated UC5822. The strain does not survive exposure to 44°C. No phage could be detected in cultures of UC5822 either before or after incubation at 44°C.

Example 7 Construction of MG4 Cultures of MG3 are grown in Lurie, broth or other complete media at 32°C until &g5Q ~ θ·5. The culture will contain approximately 5x10® cells/ml. The culture is then plated on Nutrient Agar plates supplemented with 0.2% glucose, and 0.2 % KCIO^. The plates are incubated under anerobic conditions at 32°C until colonies form (3-5 days). Growth on this media under these conditions selects for E. coli which have mutations in the chi A, B, G or D gene. See, "Expts. in Molecular Genetics, J. Miller, pps 226-227.

Mutation in chi3, C or D will not lead to the isolation of MG4. Among the mutations affecting chiA expression will be point mutations in chiA and deletions extending to the left or right of chlA. Deletions 1° extending to the left of chiA may result in disruption of the adjacent uvrB gene and thus confer a UV sensitive phenotype on the organism.. For the same reason, rightward extending deletions from the chip gene may also confer a UV sensitive phenotype on the organism. Chlorate resistant colonies obtained from the anerobic incubation are tested to determine if they are now UV sensitive.

This is conveniently done by screaking a chlorate resistant colony across a pefcri dish, covering 1/2 the dish and subjecting the other half to 10 J/m' of 254 nm UV light. This dose is sufficient to kill UV sensitive cells but not UV resistant mutants. The UV sensitive mutants (comprising mutations in ch1A or chip are tested for the presence of the defective lambda prophage. This is done by cross streaking the cells through a streak of a homoimmune phage. Lambda sensitive bacteria are killed by the phage at the cross-streak; lambda lysogens are immune to super infection and are not killed. As a consequence of the location of the ch1A and chip genes, the lamba genome and the uvrB gene, all UV sensitive chlD mutants will be lambda sensitive whereas some UV sensitive chiA mutants may be lambda lysogens. UV sensitive, lambda lysogens therefore contain deletions of chlA which extend leftward into uvr B. If the deletion extends through uvrB it may extend into the biotin operon and possibly into the structural lambda genes. Deletions of structural lambda - 22 genes have been obtained in this manner (Grier, virology 616:589-604 (1975). All UV sensitive, chi A , lambda lysogens are infected with λ virA . After 2-1/2 hours, the lysates are plated for λ virA phage and for λ vir A~ recombinants. MG4 candidates which propagate λ virA and/or produce λ virA' phages are discarded; candidates which fail to complement λ virA** or produce virA' carry a deletion extending from ch1A through the A gene of lambda. Those MG4 candidates which possess deletions from chiA through the A gene of lambda are tested for the lytic bacteria property (lysis upon growth at>38°C). Candidates containing the deletion which have retained the lytic bacteria property are purified as MG4.

Example 8 Cloning in MM294(cl857) and UC5822 E. coli strain. MM294 was incubated in the presence of λ cl 857. After overnight growth at 32°C, surviving bacteria were isolated and purified. Clones which were immune to super infection and which were unable to grow and produced phage at 44°C were isolated. This resulting cl85 7 lysogenic strain, MM294 (λ £1857) , and Ξ. coli strain UC5822 were made competent by CaCl^ treatment and transformed with pDN5, a plasmid carrying genes for the E. coli LT-B antigen and for ampicillin and tetracyline resistance.

Ampicillin and tetracycline resistant transformants of both lytic bacteria strains grew well in a standard nutrient broth at 30~32°C and expressed LT-B antigen. The bacteria were pelleted by centrifugation and transferred to a standard nutrient broth at 42°C.

Within about 90-120 minutes, cell lysis was evident and substantially complete. LT-B antigen was released into - 23 the broth. In a sample of the MM294(cl857) transformant 7 8 comprising 4x10 cells/ml, about 2x10 lambda phage were collected per ml. In a similar sample of the UC5822 transformants, no phage (<20/rnl) were collected.

Example 9 Cloning in UC5822 A seed culture of E. coli uC5822 containing the plasmid pESS2 which carries the genes for E. coli LT-B antigen was inoculated in a 5 ml tube of L broth containing ampicillin. After δ hours the tube contents were transferred to 500 ml of culture medium containing ampicillin and incubated overnight with shaking at 32°C. To inoculate 10 L, 400 ml of the overnight culture was transferred to 10 L of medium containing ampicillin in a Virtis bench-top fermentor. The culture was maintained at 32°C and each hour a 100 ml sample was monitored for growth at Α^?θ. At 4 hours the culture was fed 200 ml of 50% dextrose and 0.1 ml of an antifoam agent. At 8 hr the culture was at 4.8 Α^θ units and was shifted to 43°C. The culture was fed again at 10 hr with 200 ml of 50% dextrose, and at 14 hr the culture was stopped. A sample of a cell concentrate (pellet) and of the cell supernatant at 4 hr, δ hr, 8 hr, 9 hr, 10 hr, .6 hr and 14 hr were tested for LT„ bv using an ELISA test with known concentrations of LTO as standards.

JO The results are shown in Table I. In the first 4 to 8 hr greater than 90% of the LT-B resided in the cell, but within 2 hr after the temperature shift (10 hr after inoculation), 90% of the LT-B was in the supernatant. At 6 hr after the temperature shift, 95% of the LT-B was in the supernatant; LT-B represented 8.5% of the total protein. The yield of LT-B was far greater than the yield - 24 from Ξ. coli MM294 transformed with pESS2.

Within 2-4 hours after the temperature shift lysis was evident by increased viscosity of the culture media and visible cell debris. By 4-6 hours after the temperature shift the viscosity was greatly reduced and the culture was easily pumped through an ultrafiltration apparatus to remove all debris and any remaining unlvsed cells. The increased viscosity reflects release of high molecular weight DNA and RNA into the media; action of endogenous nucleases ultimately results in an observable decrease in viscosity.

Table Time After Inoculation nW ag/tal ug/ml Total LT A,Call Protein LT& As Per Cen of Total Cell Protein 4 4 hr h& η3(ίϊ»1 I Super 0.58 0.734 0.41 0.06 0.07X 6 hr Pellet 1.1 6 hr Super 1.8 1.19 .11 0.1X (shift; > 8 hr Pellet 4.8 1.24 3.9 (43°) 8 Super 0.29 0.3X 10 tftX* Pellec 6.2 0»646 1.2 10 hr Super 14.8 2.5Z 10.6 hie Pellet 5.0 0.652 1.8 10.6 hr Super 39.38 6.3X 14 hr Pellet 1,8 14 hr Super 4.2 0.652 53.83 8.5X - 25 Example 10 Construction of Lytic Salmonella An interspecies cross between Salmonella and MG3 is performed, either through conjugation or DNA transformation. Salmonella strains are normally tetracycline sensitive? MG3 is tetracycline resistant. Salmonella recombinants which have attained resistance to tetracycline are tested for their ability to grow at 42^C. Those Salmonella which lyse at this temperature have acquired, through recombination, the lytic bacteria function of MG3. This experiment is possible because (1) the lambda lytic functions are expressed in Salmonella and (2) sufficient homology exists between Salmonella and E„ coli (MG3) to permit recombination of the E. coli sequences which flank the genes into Salmonella.

Example 11 Construction of Lytic Bacillus Method 1. The genetic elements sufficient to direct lysis of a host include the λ c!857, N, Q, S and R genes and the promoter. The restriction maps of JLj these genes is known (Molecular Cloning, Maniatis et al., Cold Spring Harbor Laboratory, N.Y.). The genes are subcloned from lambda onto a plasmid (for example pBR322). A fragment of DNA from Bacillus is inserted into the plasmid at a site in a non-essential region. It is not necessary to characterise the nature or function of the host strain DNA or its orientation in the plasmid.

The Bacillus is then incubated with the purified plasmid DNA, and antibiotic resistant tr ansforman ts are selected. These transformants are tested to determine if they lyse after exposure to high temperature. In these transformants, the plasmid containing the lambda genes is - 26 1 present as an autonomously replicating unit or is integrated into the host chromosome through a recombination event between homologous Bacillus DNA on the plasmid and on the chromosome. The integration of DNA carried by plasmids which cannot replicate into the Bacillus chromosome has been described (Haldenway et al., J. Bact. 142:90-98, 1980). This method requires the expression of the lambda lytic genes in the recipient host, but does not require homology between the B. coli sequences of MG 3 and the recipient bacteria.

Method 2. The phage phi-105 infects Bacillus, is temperate, and has a mutatable cI-like repressor.Derivatives of phi-105 can be made which have temperature sensitive mutations affecting this repression. Phage derivatives which lack excision or replication functions can be isolated through mutagenesis or by isolation of deletion strains. (Flock, Mol. Gen. Genet, 155:241-247, 1977). A phage derivative is obtained which possesses a thermo-labile repressor. A lysogen of this mutant is made by infecting sensitive cells with the phage at 30°C, isolating surviving cells and testing these cells for immunity to super infecting phage and for inability to grow at 40°C. Such an organism is a phage-producing lytic bacteria. To isolate a defective lytic bacteria, the bacteria is mutagenized and surviving colonies are replica plated onto an undeveloped lawn of phi-105 sensitive Bacillus. Temperature-sensitive colonies which, following exposure to high temperature, produce few or no phage on these lawns, contain, mutations affecting phage propagation. More stringent mutants may be obtained by repeating the mutagenesis. In order to mobilize this construction and easily select for transfer of this construction it is preferable to isolate a derivative which harbours an antibiotic resistance marker - 27 linked to the phi-105 genome. This can be accomplished by cloning random fragments of the Bacillus chromosome into a plasmid (which is incapable of replication in Bacillus) which carries an antibiotic resistance determinant (such as pBR322). Transformed? drug resistant cells are isolated which contain the integrated plasmid. In some of these cells the plasmid will have integrated near to the site of phi-105. The unfractionated pool of drug resistant colonies is infected with the generalized Bacillus transducing phage, pBSl. Stock of pBSl, which functions in Bacillus in exactly the same manner as PlcmlOO functions in E. coli, is used to transduce Bacillus cells to drug resistance. These drug resistant transductants are tested to determine if they are thermosensitive, lytic bacteria. Approximately 1% of transductants will have acquired the lytic bacteria proper ties.

The preceding disclosure and examples show that the methods and compositions of matter of the invention are useful to produce and externalize products in bacteria. While the preferred embodiments of the invention are illustrated by the above, the invention is not limited to the precise constructions disclosed herein but, rather, includes all embodiments and modifications coming within the scope of the following claims.

Claims (40)

1. CLAlRflS

1. A method of producing a gene product which comprises (i) culturing a temperature sensitive bacteria, which bacteria*. (a) expresses the gene product intracellularly, (b) is a lysogen defective in excision and replication functions and 5 (c) contains within the prophage DNA sequence a temperature sensitive phage repressor gene and functional phage lysozyme-encoding genes, under permissive conditions such that the gene product is expressed intracellularly and the lysozyme-encoding genes are repressed and then (ii) raising the temperature to produce restrictive conditions 10 such that the lysozyme-encoding genes are expressed.

2. The method of claim 1 wherein the temperature-sensitive phage repressor gene is a temperature sensitive lambda cl gene. 15

3. The method of claim 2 wherein the cl gene is the £1857 mutant.

4. The method of claim 2 wherein the bacteria is an E. coli lambda lysogen. 2o 5. The method of claim 4 wherein the bacteria is E. coli strain UC5822 which is constructed by infecting strain N99 (E. coli K12 qalK lac Z su° thi) with phage λ int6 red3 cl857 £80 and λ hy5 cl irnm 21 Δ b2.

5. The method of claim 4 wherein the lambda prophage DNA sequence is 25 deleted in the genes lying between map positions 58 and 71 and is mutated in the 0 and P genes resulting in loss of 0 and £ gene functions.

6. 7. The method of claim 6 wherein the lambda prophage DNA sequence is flanked by a selectable marker which is a gene coding for a selectable -,'j trait and the 0 and £ mutations are point mutations.

7. 8. The method of claim 7 wherein the lambda prophage DNA sequence is flanked on the upstream end by the tnlO transposable tetracycline resistance element and the 0 and £ genes are the 029 and £3 genes. -309. The method of claim 7 wherein the bacteria is derived from £. col 1 strain MGO which is constructed by infecting strain C600 (£. coli Sull\ K12, galK. lacZ. sull, thi) with phage λ £1857, £3, 029.

8. 10. The method of claim 7 wherein the bacteria is strain MG 3 derived from N99 and having the genotype N99 (λΔ 58-71, c1857, £3, 029).

9. 11. The method of claim δ wherein the lambda prophage DNA sequence is deleted in the genes lying between map positions 3 and 71.

10. 12. The method of claim 11 wherein the lambda prophage DNA sequence is flanked by a selectable marker which is a gene coding for a selectable trait and the 0 and £ mutations are point mutations.

11. 13. The method of claim 12 wherein the lambda prophage DNA sequence is flanked on the upstream end by the tnlO transposable tetracycline resistance element and the 0 and £ genes are the 029 and £3 genes.

12. 14. The method of claim 12 wherein the bacteria is an £. coli MG4 strain derived from N99 and having the genotype MG4 [N99 (λΔ3-71, eI857 £3, 029) galK. lacZ. thi, gal::tnlO tet^].

13. 15. A DNA fragment comprising (i) a selectable marker which is a gene coding for a selectable trait; (ii) a lambda prophage DNA sequence having a temperature sensitive pi repressor gene and functional lysozyme encoding genes such that the lysozyme-encoding genes are repressed under permissive conditions and expressed under restrictive conditions, wherein the prophage DNA sequence includes functional £, 0, £ and R genes, is substantially deleted in the genes lying between map positions 58 and 71 and has mutations in the 0 and £ genes resulting in loss of 0 and £ gene functions; and (iii) flanking DNA sequences homologous to a contiguous sequence in the chromosome of a host bacterial cell to permit recombination between the fragment and the host cell chromosome to occur.

14. 16. The DNA fragment of claim 15 wherein the cl gene is the cI857 gene, the 0 and £ mutants are the 029 and £3 mutants and the selectable marker is the tnlO transposable tetracycline resistance element. -3117. The DNA fragment of claim 15 wherein the prophage DNA sequence is substantially deleted in the genes lying between map positions 3 and 71.

15. 18. The DNA fragment of claim 17 wherein the cl. gene is the cI857 gene, the 0 and P. mutants are the 029 and P3 mutants and the selectable marker is the tnlO transposable tetracycline resistance element.

16. 19. A bacteria comprising the DNA fragment of claim 15.

17. 20. A bacteria comprising the DNA fragment of claim 17.

18. 21. The bacteria of claim 19 which is an E., coli.

19. 22. The bacteria of claim 20 which is an E.. coli.

20. 23. The bacteria of claim 21 which is derived from strain MGO [E.. col i strain C600 (E. coli Sul1 + , K12, galK. lacZ, sull, thi) infected with λ CI857 P3 029].

21. 24. The bacteria of claim 21 which is strain MG3 [N99 (λΔ 58-71, CI857.P3, 029)].

22. 25. The bacteria of claim 22 which is strain MG4 [N99 (λΔ 3-71, cl857.£3, 029) galK. lacZ. thi, gal,’tnlO tet R ].

23. 26. A method of making a temperature sensitive bacteria which comprises transforming a bacteria with a DNA fragment comprising (i) a selectable marker which is a gene coding for a selectabe trait; (ii) a lambda prophage ONA sequence having a temperature sensitive cl repressor gene and functional lysozyme encoding genes such that the lysozyme-encoding genes are repressed under permissive conditions and expressed under restrictive conditions, wherein the prophage DNA sequence includes functional N, Q, S. and R genes, is substantially deleted in the genes lying between map positions 58 and 71 and has mutations in the 0 and P. genes resulting in loss of 0 and P gene functions; and, (i 1 i) flanking DNA sequences homologous to a contiguous sequence in the chromosome of a host bacterial cell to permit recombination between the fragment and the host cell chromosome to occur. -3227. The method of claim 26 wherein the cl gene is the cl857 gene, the 0 and £ mutants are the 029 and P3 mutants and the selectable marker is the tnlO transposable tetracycline resistance element.

24. 28. The method of claim 26 wherein the prophage DNA sequence is substantially deleted in the genes lying between map positions 3 and 71.

25. 29. The method of claim 28 wherein the cl gene is the £1857 gene, the 0 and P. mutants are the 029 and P3 mutants and the selectable marker is the tnlO transposable tetracycline resistance element.

26. 30. The method of claim 26 wherein the bacteria is an £. coli.

27. 31. The method of claim 28 wherein the bacteria is an E. coli.

28. 32. A method of making a lytic bacteria which comprises transforming a bacteria with the DNA fragment of any claims 15 through 18.

29. 33. The method of claim 32 wherein the bacteria is £. coli.

30. 34. A method of producing a product in a bacteria which produces or is made to produce the product which comprises transforming the bacteria with the DNA fragment of any of claims 15 through 18; culturing the transformed bacteria under permissive conditions such that the product is made; changing the temperature to provide restrictive conditions; and, optionally, recovering the product from the culture medium or a concentration thereof.

31. 35. A method of producing a product in a bacteria which produces or is made to produce the product which comprises integrating the DNA fragment of any of claims 15 through 18 into the chromosome of the bacteria, culturing the transformed bacteria under permissive conditions such that the polypeptide is expressed; changing the temperature to provide restrictive conditions; and, optionally, recovering the polypeptide from the culture medium or a concentration thereof.

32. 36. A method of producing a gene product as claimed in any of claims 1 to 14 substantially as hereinbefore described with reference to the examples

33. 37. A gene product whenever prepared by a method as claimed in any of claims 1 to 14 or 3fc. -3338. A DSMA fragment as claimed in any of claims 15 to 18 substantially as hereinbefore described with reference to the examples.

34. 39. A bacteria comprising a DNA fragment as claimed in claim 38.

35. 40. A method of making a temperature sensitive bacteria as claimed in any of claims 26 to 31 substantially as hereinbefore described with reference to the examples.

36. 41.. A temperature sensitive bacteria whenever prepared by a method as claimed in any of claims 26 to 31 or claim 40.

37. 42- A method of making a lytic bacteria as claimed in claim 32 substantially as hereinbefore described with reference to the examples.

38. 43- , A lytic bacteria whenever prepared by a method as claimed in claim 32 or claim 42.

39. 44. A method of producing a product in a bacteria as claimed in claim 34 or 35 substantially as hereinbefore described with reference to the examples,

40. 45. A product produced by a method as.claimed in claim 34 9 claim 35 or claim 44„